Hsp90b1 knockout targeted to male germline: a mouse model for globozoospermia  Christophe Audouard, M.S., Elisabeth Christians, Ph.D.  Fertility and Sterility  Volume 95, Issue 4, Pages 1475-1477.e4 (March 2011) DOI: 10.1016/j.fertnstert.2010.12.006 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Histologic and cellular analyses of Hsp90b1-deficient spermatogenesis (see Supplemental Materials and Methods, available online at www.fertstert.org, for experimental procedures). (A) Histologic sections of wild-type (WT; panels I, III, and V) or Vasa-Cre Hsp90b1del/flox (mutant [MT]; panels II, IV, and VI) testis and WT (panel VII) or MT (panel VIII) epididymis. (A, panels I–IV) Hematoxylin and eosin (HE)–stained seminiferous tubules are shown at two different magnifications: scale bars: I and II = 100 μm; III and IV = 50 μm. (A, panels V and VI) Histologic sections stained by immunofluorescence using Hsp90b1 antibodies. Insets display higher magnification, demonstrating Hsp90b1 expression in all the cells of seminiferous tubules in WT animals (V) whereas expression is restricted to somatic–Sertoli cells in MT animals (VI). (A, panels VII and VIII) HE-stained epididymis sections show the abundant presence of spermatozoa in both WT (VII) and MT (VIII) mice. Insets display representative examples of normal sperm head and round-headed sperm, respectively. (B) The sperm heads were classified into four categories: normal, abnormal 1 (sperm head is still exhibiting hooked shape), abnormal 2 (sperm head is globular), and abnormal 3 (highly abnormal sperm head). The graph shows the percentage of each category (WT spermatozoa: n = 100; MT spermatozoa: n = 200). (C) Confocal images of Lens culinaris agglutinin (LCA)–fluorescein isothiocyanate (FITC) (glycoconjugates) and TO-PRO-3 (DNA) staining of WT (panel I) and MT spermatozoa (panels II, III, and III′). In panel III′, the contrast of the image shown in III has been enhanced to reveal the localization of LCA staining in MT spermatozoa exhibiting abnormal morphology type 3. (D) Confocal images of streptavidin-FITC (biotinylated proteins in mitochondria) and TO-PRO-3 (DNA) staining of WT (panels I and I′) and MT (2 examples of abnormal morphology: panels II/II′ and III/III′) spermatozoa. In panels I′-III′ the contrast of the images has been enhanced to reveal the localization of streptavidin-FITC staining in WT and MT spermatozoa. (E) Immunodetection of HSPA5 (ER chaperone) and TO-PRO-3 (DNA) staining of WT (panel I) and MT spermatozoa (panels II–III′). In panel III′, the contrast of the image shown in III has been enhanced to reveal the localization of HSPA5 staining in MT spermatozoa and the position of the intermediate piece. Fertility and Sterility 2011 95, 1475-1477.e4DOI: (10.1016/j.fertnstert.2010.12.006) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Testis size is normal in adult male mice whose germline is deficient in Hsp90b1. The graph shows the ratios of testis weight to body weight measured in wild-type (WT; n = 14) and mutant (MT; n = 3) mice, mean ± SD. Fertility and Sterility 2011 95, 1475-1477.e4DOI: (10.1016/j.fertnstert.2010.12.006) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Vasa-Cre Hsp90b1del/del spermatozoa are abnormal Vasa-Cre Hsp90b1del/del spermatozoa are abnormal. Sperm was collected from the cauda epididymis and fixed before observation. (A, panel I) Wild-type (WT) spermatozoon showing the typical hook-shaped head, intermediate piece, and flagellum. (A, panel II) Hsp90b1-deficient spermatozoa exhibit abnormal head and intermediate piece, as indicated by arrowhead. Bar scale (I) = 10 μm; I and II are same magnification. (B) Higher magnification showing representative examples of WT (I) and mutant (MT; II) sperm head. Images I and II are highly contrasted to make visible the intermediate piece in mutant animals. (B, panels I′ and II′) DNA (nuclear, mitochondrial) staining with Sybr Green I (1:1,000, Sigma, St. Louis, MO). (B, panels I″ and II″) DNA staining with TO-PRO-3 (1:500, Invitrogen, Carlsbad, CA). Fertility and Sterility 2011 95, 1475-1477.e4DOI: (10.1016/j.fertnstert.2010.12.006) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions